Phytochemical Content, Antibacterial Activity, and Antioxidant, Anti-Inflammatory, and Cytotoxic Effects of Traditional Medicinal Plants against Respiratory Tract Bacterial Pathogens

Respiratory tract infections (RTIs) are frequent ailments among humans and are a high burden on public health. This study aimed to determine the in vitro antibacterial, anti-inflammatory, and cytotoxic effects of indigenous medicinal plants used in the treatment of RTIs, namely, Senna petersiana, Gardenia volkensii, Acacia senegal, and Clerodendrum glabrum. Dried leaves were extracted using various organic solvents. Antibacterial activity was quantified using the microbroth dilution assay. Protein denaturation assays were used to evaluate anti-inflammatory activity. The cytotoxicity of the extracts towards THP-1 macrophages was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Antioxidant activity was determined using free radical scavenging activity and ferric-reducing power. Total polyphenolics were quantified. Liquid chromatography mass spectrometry was used to evaluate the acetone plant extracts. Nonpolar extracts had noteworthy antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium smegmatis where MIC values ranged between 0.16 and 0.63 mg/mL. At 100 μg/mL, A. senegal, G. volkensii, and S. petersiana had a nonsignificant effect on the viability of the THP-1 macrophages. The LC-MS analysis of the leaf extracts of S. petersiana detected Columnidin, Hercynine, L-Lysine citrate, and Gamma-Linolenate. A pentacyclic triterpenoid, cochalate, was detected in G. volkensii. Two flavonoids 7-hydroxy-2-(4-methoxyphenyl)-4-oxo-chroman-5-olate and (3R)-3-(2,4-dimethoxyphenyl)-7-hydroxy-4-oxo-chroman-5-olate were detected in the C. glabrum extract. The findings from this study indicated that the leaves of the selected plant extracts possess antioxidant, anti-inflammatory, and antibacterial activity. Therefore, they may serve as good candidates for further pharmaceutical investigations.


Introduction
Respiratory tract infections (RTIs) are frequent ailments among humans and are a high burden on public health. Te efects of RTIs are most prevalent in developing countries in sub-Saharan Africa, where higher economic losses, morbidity, hospital admissions, and mortality rates are experienced [1]. Respiratory tract infections can be categorized into upper and lower tract infections and may be caused by bacteria, viruses, fungi, or parasites [1]. Te majority of RTIs are transmitted mainly through inhalation of infected aerosols or contact with bodily fuids [1]. Lower respiratory tract infections (LRTIs) were counted among the top fve causes of death in children younger than 5 years [1,2]. Various opportunistic bacteria, including Streptococcus pneumoniae, Haemophilus infuenzae, Klebsiella pneumoniae, Staphylococcus aureus, Acinetobacter species, Streptococcus viridans, Pseudomonas aeruginosa, Escherichia coli, and Proteus species have been identifed as common causes of LRTIs [1][2][3].
Microbial infections have been associated with the upregulation of proinfammatory signal molecules and the release of lysosomal enzymes which cause tissue damage during prolonged infammation [4]. Free radicals such as reactive oxygen species (ROS) contribute to the development of infammatory diseases. Chemical species known as antioxidants are capable of scavenging ROS and have been linked to the remediation of infammatory disorders [5]. Synthetic drugs such as rifampicin, erythromycin, benzylpenicillin, chloramphenicol, and tetracycline are used for the treatment of respiratory bacterial pathogens [6]. Te drawback of conventional antibiotics is their associated side efects which include nausea, vomiting, diarrhoea, abdominal pain, loss of appetite, bloating, dizziness, and loss of appetite [7].
Medicinal plants have been the cornerstone of herbal medicine, whereby infusions, macerations, tinctures, and decoctions of diferent plant parts have been used to treat diseases [8]. Te success of medicinal plants as an alternative source of therapy for humans stems from their ability to synthesize bioactive phytochemicals such as phlobatannins, anthocyanin, betacynin, favonoids, tannins, phenolics, alkaloids, glycosides favonoids, steroids, saponins, and terpenoids [9]. Phytochemicals play critical protective roles against pathogenic infections commonly caused by bacteria, fungi, viruses, parasites, herbivores, insects, pests, and harsh environmental conditions such as unfavourable climate and insufcient nutrient supply [10].
Bioactive phytochemicals in medicinal plant extracts have various structural complexities and may exert their therapeutic efects by acting on new targets [11]. Moreover, the additive and synergistic efects associated with phytochemicals may further provide a multitargeted therapeutic approach that might be more advantageous than singletarget-based drugs [12]. Herbal medicine, a prominent form of traditional medicine, serves as a relief for populations in developing countries where health facilities such as hospitals, dispensaries, pharmacies, and clinics are either underdeveloped or expensive for patients [13][14][15].
Participatory collaboration with indigenous cultures plays a pivotal role in retrieving anecdotal information, identifcation, and documentation of medicinal plants used to treat various diseases [16]. Sub-Saharan Africa boasts an estimated 45, 000 plant species [17], of which 45.5% (20 456) are indigenous plant taxa spread across the Republic of South Africa. Only 10% (≈2062 plant species) of the total fora of South Africa has been recorded to be used in traditional medicine [18]. It has been reported that about 27 million of the South African population depend on traditional medicine [19].
Gardenia volkensii belongs to the Rubiaceae family and has common names such as bushveld gardenia, sand veld gardenia, savanna gardenia, and Transvaal gardenia [20]. Tis plant has hairless obovate leaves that are grouped at the tips of the short lateral twigs. Te fowers are axillary, solitary, large, and showy in white. G. volkensii grows in warm temperate regions which include Africa, Madagascar, East and Southeast Asia, the western Pacifc, and Hawaiian islands [21]. G. volkensii has a wide distribution in Africa in countries such as Angola, Botswana, Eswatini, Ethiopia, Kenya, Malawi, Mozambique, Namibia, Tanzania, Uganda, Somalia, South Africa, Zambia, and Zimbabwe. Tese countries represent 71.4% of countries in which G. volkensii is indigenous [20]. Te fruits and roots of G. volkensii are widely used in ethnomedicine for the treatment of asthma, infertility, earache, sore eyes, epilepsy, headache [22], asthma, chest complaints, colds, pneumonia, sore throat, and tuberculosis [20]. Te nutritional value of the fruits of G. volkensii is indicated by their consumption by various animals such as elephants, kudu, velvet monkeys, and baboons [22]. Senna petersiana belongs to the Fabaceae family and can grow to a height of 7 m with dark green leaves which have several leafets facing opposite. Te fowers are large and yellow, while the pods produced are green and fattened. Seventy-four percent (74%) of Senna species are largely distributed in America; Australia has about 13%; and Africa together with Madagascar constitutes 10% of this genus distribution [23]. S. petersiana leaves are traditionally used to treat malaria and typhoid fever, and the roots are used for the treatment of coughs, colds, syphilis, and helminthic infections [24]. Te pods produced by S. petersiana have been reported to be consumed either fresh or cooked [24]. Acacia senegal belongs to the Fabaceae family and is a thorny shrub tree of 2-6 or even 12 meters high with very branched and ascending branches. Its leaves are green-grey, alternating, and bipinnate. Te cream-coloured small fowers produce pods that are straight and slightly curved [25]. A. senegal is a drought-resistant species that commonly occurs in arid, semi-arid, and subtropical regions in sub-Saharan Africa such as Senegal, Cameroon, and Sudan [26,27]. Te distribution of A. senegal extends to the Arabian Peninsula, Pakistan, and India [28]. A. senegal is famous for its gum arabic, a dried exudate rich in soluble fbres, which emerges from slits made in the bark of the stems and branches [28]. Te diferent parts of A. senegal can be used in the form of decoctions, macerations, or administered dry. Tis species is traditionally used for the treatment of colds, coughs, diarrhoea, dysentery, expectorant, gonorrhoea, sore throat, and disorders of the urinary tract [25,27]. Carissa bispinosa is an evergreen densely bushed small tree with a height of up to 1-5 meters that belongs to the Apocynaceae family. Te branched stem of this species is green in colour, spiny, and rich in white latex. Te leaves are ovate/rounded and are positioned in an opposite-decussate arrangement. It produces small white/ tinged pink fragrant fowers, and the fully ripened fruits are red in colour and ovoid in shape. Species of the Carissa genus are native to Africa, Indo-China, Australia, and New Caledonia [29]. Te roots of C. bispinosa are extracted and drunk as cough and diarrhoea medicine [30]. Local people in KwaZulu natal, South Africa, depend on the sale of fruits from Carissa spp. Tese fruits would usually be sold in the summer months. Te popularity of fruits is attributed to their nutritional content such as vitamin C, calcium, magnesium, and phosphorus [31]. Although the antioxidant activity of favonoids from the fruit of C. bispinosa has 2 Evidence-Based Complementary and Alternative Medicine previously been reported [32], there is little scientifc documentation of the biological activities of the leaves of this plant. Clerodendrum glabrum belongs to the Lamiaceae family. Its common names include glory bower, bag fower, and bleeding heart [33]. Te roots and the bark are usually dried, pulverised, and then boiled to treat ailments such as oral ulcers, diarrhoea, and coughs [34]. C. glabrum is widely distributed in Asia, Australia, America, and Africa [35].
Tere are a few to no reports of the biological activities of the plant species selected for this study, despite their wide traditional use. Te focus of this study was to select underexplored traditionally used medicinal plants which may hold potential for the development of efcacious antibacterial RTIs caused by common pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. Staphylococcus aureus causes bacteremia, endocarditis, skin and soft tissue infections, osteoarticular infections, joint infections, and pleuropulmonary infections [36]. Pseudomonas aeruginosa has been reported in bacteremia, ventilator-associated pneumonia, urinary and respiratory tract infections, and skin and soft tissue infections [37]. E. coli is the most frequent cause of bloodstream and urinary tract infections among Gramnegative bacteria [38]. We determined the antibacterial, antioxidant, anti-infammatory, and cytotoxic efects of the selected medicinal plants and showed the potential the active extracts have for further studies involving the treatment of bacterial RTIs and their accompanying symptoms.  Table 1 shows the selected plants and their traditional uses. Te plants were collected at the Lowveld National Botanical garden located in Mbombela, Mpumalanga, South Africa. Te identities of the plants were verifed by botanist Dr. Bronwyn Egan from the Larry Leach herbarium at the University of Limpopo, and subsequently, voucher specimens were prepared and deposited in the herbarium. Te leaves were dried at ambient temperature. Once dried, the plant material was ground to a fne powder using a commercial blender. Te powdered material was stored in airtight glass bottles in closed cabinet compartments, away from sunlight.

Plant Extract Preparation.
One gram of each of the ground plant materials from the selected plants was extracted with 10 mL of n-hexane, dichloromethane, acetone, and methanol in diferent 50 mL polyester centrifuge tubes. Te tubes were shaken at 200 rpm in a shaking incubator for 30 mins. Decoctions of the plant leaves were prepared by boiling 1 g of ground leaves in 10 mL distilled water for 5 min. Te extracts were fltered from the plant material into glass vials. Te solvents were evaporated under a stream of air at ambient temperature. Te extracts were quantifed and reconstituted to a concentration of 10 mg/mL using acetone to prepare stock solutions.

Broth Microdilution
Assay. Te broth microdilution assay described by Elof [40] was used. Te antimicrobial activity of the plant extracts was evaluated using E. coli (2 × 10 10 cfu/mL), S. aureus (2 × 10 8 cfu/mL), M. smegmatis (2 × 10 5 cfu/mL), M. tuberculosis (2 × 10 5 cfu/mL), and Evidence-Based Complementary and Alternative Medicine Evidence-Based Complementary and Alternative Medicine Pseudomonas aeruginosa (3 × 10 9 cfu/mL) as test microorganisms. Te extracts were reconstituted to a concentration of 10 mg/mL using acetone. Sterile distilled water (100 μL) was added to each well of a 96-well microtitre plate. Te extracts (100 μL) were serially diluted with distilled water in the 96-well microtitre plates to achieve a concentration of 0.02-2.5 mg/mL. Each microorganism culture (100 μL) was separately added to each well aseptically. Rifampicin was used as a positive control for antimycobacterial activity, and tetracycline was used as a positive control for antibacterial activity. Sterile distilled water was used as the negative control. Te microtitre plates were incubated for 24 h at 37°C for E. coli, S. aureus, M. smegmatis, and P. aeruginosa. After incubation, 40 μL of 0.2 mg/mL of p-iodonitrotetrazolium chloride (INT) dissolved in sterile distilled water was added to each well and further incubated for 30 min. Te plates inoculated with M. tuberculosis were incubated for 5 days after adding to the extracts and after incubation, 20 μL of 0.02 mg/mL resazurin dissolved in distilled water was added. Te plates were incubated for 4 h for optimal colour development. Te minimum inhibitory concentrations (MIC) were determined as the lowest concentration of the plant extract that was able to inhibit bacterial growth. Total antibacterial activity was calculated as the total mass (mg) of the extract divided by the MIC value (mg/mL). Total activity (TA) is expressed as mL and represents the volume to which the extract (obtained from 1 g of plant material) can be diluted and still maintain efective antibacterial activity [41].

Minimum Bactericidal Concentration Determination.
Te procedure described by Senhaji et al. [42] to determine the minimum bactericidal concentrations (MBC) of the extracts was performed with some modifcations. Briefy, the microtitre plates used for determining MIC values were incubated for an additional 24 h to make a cumulative 48 h of incubation. Te inability of the microorganisms to grow was indicated by the clear wells and 10 μL of the samples were subcultured on nutrient agar and incubated for 24 h at 37°C. MBC was determined to be the lowest concentration that showed no bacterial growth on the nutrient agar plates.

Free Radical (DPPH) Scavenging
Assay. Te free radical scavenging activity of the plant extracts was quantifed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method reported by Chigayo et al. [43]. Diferent concentrations of the extracts (15.63-250 μg/mL) were prepared in a volume of 1 mL of solution. L-ascorbic acid was used as a positive control by preparing it in the same concentration range as the extracts. To these 1 mL solutions, 2 mL of 78.5 μg/mL DPPH solution were added and vortexed thoroughly. All the prepared mixtures were left to stand in the dark for 30 min. Te control solution was prepared by adding 2 mL of 78.5 μg/mL DPPH to 1 mL of distilled water. After the elapsed time, the solutions were analysed with a UV/VIS spectrophotometer. Te absorbance of the solutions was read at 517 nm.
%scavenging activity � (Ac − As) Ac where Ac is the absorbance of the control solution and As is the absorbance of the extracts.

Ferric-Reducing Power
Assay. Te ferric-reducing power of extracts was determined by using the procedure detailed by Vijayalakshmi and Ruckmani [44] which measures the formation of Pearl's Prussian blue at 700 nm. Varying concentrations of the extracts (39-625 μg/mL) were prepared in 2.5 mL aliquots in test tubes and mixed with 2.5 mL sodium phosphate bufer (0.2 M, pH 6.6), followed by 2.5 mL of 1% aqueous potassium ferricyanide. Te mixtures were vortexed after the addition of solutions. Te mixtures were incubated at 50°C for 20 min. Two millilitres of 10% w/v aqueous trichloroacetic acid were added to the test tubes after incubation. Te mixtures were centrifuged at 3000 rpm for 10 min, and 5 mL of the resulting supernatant was transferred to clean test tubes. To these solutions, 5 mL of distilled water and 1 mL of 0, 1% aqueous ferric chloride were added consecutively, with thorough mixing after each addition. A UV/VIS spectrophotometer was used to read the absorbance of solutions at 700 nm wavelength. Te blank for this procedure was prepared in the same manner; however, the extracts were replaced by an equal amount of distilled water. L-ascorbic acid was used as a standard.

Anti-Infammatory Activity
where Ac is the absorbance of the control solution and As is the absorbance of the extracts.

Egg Albumin Denaturation Inhibition Assay.
Te reaction mixture (5 mL) consisted of 0.2 mL of egg albumin (from fresh hen's egg), 2.8 mL of 0.05 M tris-bufered saline (pH 6.4), and 2 mL of varying concentrations (0.25-2 mg/ mL) of the plant extracts and standard drug (diclofenac sodium) (1, 0.5, 0.25 mg/mL). Te product (negative) control solution (5 mL) consisted of 3 mL of 0.05 M Tris-bufered saline and 2 mL of each extract solution to consider the colour of the extracts when using the UV/Vis spectrophotometer. Egg albumin solution (0.2 mL and 4.8 mL trisbufered saline) was used as the test solution (positive control). Te positive control represented 100% protein denaturation. Te mixtures were incubated at 37 ± 2°C in a biochemical oxygen demand (BOD) incubator for 15 min and then heated at 70°C for 5 min. After heating, the solutions were allowed to cool to room temperature for 30 min. After cooling, their absorbance was measured at 660 nm by using the tris-bufered saline as a blank [46]. Te percentage inhibition of protein denaturation was calculated by using the following formula: where Ac is the absorbance of the control solution and As is the absorbance of the extracts.

Cell Viability Assay.
Te THP-1 cell line was cultured in Royal Park Medical Institute-1640 (RPMI-1640) medium, supplemented with 10% fetal bovine serum (FBS) and incubated in a 5% CO 2 /95% air fully humidifed atmosphere at 37°C. THP-1 monocytes were diferentiated into macrophages by exposure to phorbol 12-myristate 13acetate (PMA) [47]. Te diferentiation of the THP-1 cell line with PMA treatment has been previously reported to be optimal when incubated for 2-5 days [48]. It is vital to arrest the diferentiation of the cell line after 48 hr treatment with PMA to increase macrophage markers [47]. Growing THP-1 monocyte culture was diluted to 2 × 10 5 cell/mL in a 50 mL vial and pretreated with 25 μL of 100 μg/mL PMA to yield a fnal concentration of 50 ng/μL in the 50 mL vial, and the cells were seeded at 2 × 10 5 cell/ mL (per well) in fat bottom 96-well plates for 72 h in 5% CO 2 at 37°C to induce maturation of the monocytes into macrophage-like adherent cells [49]. THP-1 cells per well were seeded in 96-well plates to a fnal volume of 100 μL.
After treatment with PMA, the spent media was removed, the cells were washed with prewarmed 1X PBS, and fresh media was added followed by 24 h incubation [44]. For maintenance of the Vero cell lines, a confuent Vero cell monolayer with a cell density of 4 × 10 4 cells/well (100 μL) was seeded in 96-well plates and incubated at 37°C in a 5% CO 2 incubator for 24 h.
Extract concentrations were prepared separately; briefy, each extract was presolubilized in dimethyl sulfoxide (DMSO) to give a stock solution of 200 mg/mL. Te extracts were diluted with complete RPMI 40 or DMEM media to give fnal concentrations of 1000, 500, and 100 μg/mL. Actinomycin D was used as a positive control. For the THP-1 macrophages, the culture medium was replaced by a new complete RPMI medium containing plant extracts with the concentrations, and DMEM medium containing plant extracts with the concentrations was used for the Vero cell monolayer. Te plates were incubated for another 24 h at 37°C in a 5% CO 2 incubator, and the cell viability was evaluated using the MTT colourimetric assay. A 20 μL of MTT solution 5 mg/mL in flter-sterilised phosphatebufered saline (PBS) was pipetted into each well followed by a 4 h incubation period at 37°C in the 5% CO 2 incubator until purple precipitates were visible under a microscope. Te medium together with MTT was aspirated of the wells, and 100 μL of DMSO was added to the wells to dissolve the formazan. Te absorbance was determined at 570 nm using a microplate reader. Te absorbance of wells flled with media alone was used as a blank. Results were obtained from three independent experiments, and duplicate assays were performed for each experiment [50].

Phytochemical Study of Plant Extracts
Determination of Phenolic Content. Te Folin-Ciocalteu reagent method described by Tambe and Bhambar [51] was adopted. Ten microliters of 10 mg/mL plant extracts were diluted with 490 μL of distilled water, followed by the addition of 0.25 mL of Folin-Ciocaltleu reagent in each test tube. To this solution, 1.25 mL (7%) aqueous sodium carbonate (Na 2 CO 3 ) was added, and the mixtures were incubated in the dark at ambient temperature for 30 min. Te absorbance of the sample mixtures was measured at 725 nm. A blank was prepared similarly. Various concentrations of gallic acid (0.08-1.25 mg/mL) were prepared for the standard curve.

Total Tannin Content Determination.
To quantify the tannin content, the Folin-Ciocalteu method described by Tambe and Bhambar [51] was used. Briefy, 100 μL of 10 mg/ mL extracts were added to a clean test tube containing 7.5 mL of distilled water. Te Folin-Ciocalteu reagent (0.5 mL) was added to the mixture and vortexed thoroughly. Ten millilitres (10 mL) of a 35% solution of sodium carbonate were added to the mixture. Te mixture in the tube was transferred to a 10 mL volumetric fask, and the volume of the mixture was made up to 10 mL with distilled water. Te mixture was shaken and kept at ambient temperature for 30 min in the dark. Gallic acid was used as a standard, and reference standard solutions (0.625-1 mg/mL) were prepared. Te absorbance of the solutions was measured at 725 nm against a blank reagent blank. Tannin content was expressed as milligram gallic acid equivalence/Gram of extract (mg GAE/g). All the measurements were evaluated in triplicate. [51] was used to quantify total favonoid content. Briefy, 100 μL of 10 mg/mL of plant extracts were added to 4.9 mL of distilled water in a clean test tube followed by the addition of 300 μL of 5% sodium nitrite dissolved in distilled water. Te mixture was allowed to react at ambient temperature for 5 min. Tree hundred microliters (300 μL) of 10% aluminium chloride (dissolved in distilled water) were added to the reaction mixture. Te reaction was allowed to proceed for 5 min, after which 2 mL of 1 M sodium hydroxide was added. To construct a standard curve, a concentration gradient (16-125 μg/mL) of quercetin was prepared, and each concentration was reacted in the same manner as the extracts. Te absorbance of the samples was read against a reagent blank at a wavelength of 510 nm. Te results are expressed in milligram quercetin equivalence/ gram of extract (mg QE/g).

Quantifcation of Total Alkaloid Content.
A procedure detailed by Rao et al. [52] was followed. Working solutions of 1 mg/mL of each plant extract were prepared using dimethyl sulfoxide (DMSO). One milliliter (1 mL) of 2 M HCl was added to 1 mL of DMSO-dissolved extracts, and the resulting mixture was fltered using flter paper. Te fltrate was transferred to a 250 mL separating funnel, and to this solution, 5 mL of 0.1% bromocresol green (dissolved in methanol) was added followed by 5 mL of phosphate bufer (pH 6.6). Chloroform (1 mL) was added into the separating funnel, and the mixture was vigorously shaken, after which the funnel was allowed to stand to allow the mixture to separate into diferent layers. Te lower layer was collected in a 10 mL volumetric fask. Te process was repeated with 2, 3, and 4 mL of chloroform. Atropine was used to construct a standard curve using a concentration range of 0.0625-1.0 mg/mL. Te absorbance of the sample and standard solutions was recorded at a wavelength of 470 nm against a reagent blank. Te total alkaloid content was expressed as milligram atropine equivalent/gram of extract (mg AE/g).

Quantifcation of Total Flavonol Content.
Te aluminium chloride method detailed by Iqbal et al. [53] was followed to determine total favonol content. Plant extracts were made to 1 mg/mL, and 0.5 mL of these solutions were added to test tubes followed by the addition of 0.5 mL of 2% aluminium chloride and 1.5 mL of 5% of sodium acetate. Te solution was mixed well using a vortex. Te solution was then transferred to a 2 mL Eppendorf tube and centrifuged for 20 min to obtain a clear solution. Te absorbance of the solutions was recorded at a wavelength of 440 nm against a blank. Quercetin was used as a standard, and diferent concentrations (16-250 μg/mL) were prepared. Te results were expressed as mg quercetin equivalent per gram of extract (mg QE/g).

Quantifcation of Total Proanthocyanidin Content.
Total proanthocyanidin content was quantifed by following the procedure described by Sun et al. [54]. In a test tube, 0.5 mL of 1 mg/mL of plant extracts were prepared. To this extract, 3 mL of 4% vanillin dissolved in methanol was added, followed by 1.5 mL of 37% HCl. Te mixture was vortexed thoroughly and allowed to stand for 15 mins at ambient temperature. Te absorbance of the solutions was measured at a wavelength of 500 nm against a reagent blank. Gallic acid was used as a standard, and concentrations of 16-250 μg/mL were used to construct a standard curve. Total proanthocyanidin content was expressed as milligram gallic acid equivalence/gram of extract (mg GAE/g).

Liquid Chromatography-Mass Spectrometry (LC-MS)
Studies. Te LC-MS/MS analysis was carried out using a Waters Synapt G2 qTOF mass spectrometer. Te Synapt G2 qTOF from Waters (Milford, USA) is a high-resolution quadrupole time-of-fight (qTOF) mass spectrometer capable of data independent analysis (DIA) using Waters ms E technology. When linked to a Waters Acquity UPLC, the system can achieve good chromatographic separation between compounds followed by simultaneous acquisition of both fragmented and unfragmented mass spectra of all compounds within each peak eluting of the column, together with UV spectra produced by the photodiode array (PDA) detector placed upstream of the qTOF. Te acetone extracts were centrifuged at 12,000 rpm for 10 min before analysis. A waters HSS T3 column, 2.1 × 150 mm was used in obtaining the separation of the phytoconstituents. Two mobile phases (A) and (B) were used, where (A) consisted of 0.1% formic acid in water and (B) had acetonitrile 5 mM ammonium formate. A 5 μl volume of the extracts was injected into the analytical column for analysis. Te sample fow rate was set at 0.4 mL/min. Te MS spectra were acquired in the positive ion mode. Te mass fragmentations were identifed by using a spectrum database for organic compounds.

Statistical Analysis.
Te results were expressed as means ± standard deviation of triplicate determinations. Statistical analysis was performed by the IBM Statistical Package for the Social Sciences (SPSS) (version 22) using a two-way analysis of variance (ANOVA) followed by the Tukey multiple comparison post hoc test. Chemical structure and other parameters for each compound were searched using online database software (https://www.chemspider. com and https://www.pubchem.ncbi.nlm.nih.gov). Te Xcalibur 2.2 software (Termo Fisher Scientifc, USA) was used for data acquisition and analysis.

Results and Discussion
3.1. Extraction Yield. Secondary metabolites from plants are structurally and chemically diverse; thus, various organic solvents were used as extractants to allow maximal extraction of diferent types of important bioactive phytochemicals with varying ranges of polarities. It was observed that percentage yields of extraction depended on the polarity indices of the organic solvents. Similar observations were made by Abdisa et al. [55]. Water and methanol extracted Evidence-Based Complementary and Alternative Medicine had higher percentage yields compared to hexane, dichloromethane (DCM), and acetone. Tis suggested that the plant material possessed more polar compounds than nonpolar ones.

Antibacterial Activity.
Te lower the concentration of an extract required to inhibit bacterial growth, the greater the antibacterial activity it possesses. Noteworthy activity was regarded as the concentration of the extracts showing activity at minimum inhibitory concentrations (MIC) less than 1 mg/ mL. Some of the antibacterial activity shown by the various extracts was comparable to that of tetracycline ( Table 2). Tetracyclines are a class of antibiotics with a broad-spectrum activity against numerous Gram-positive and Gram-negative bacteria by inhibiting protein synthesis [56]. Te acetone leaves extracts of C. bispinosa, S. peterisana, G. volkensii, and C. glabrum had broad-spectrum antibacterial activities against S. aureus, E. coli, P. aeruginosa, and M. smegmatis with MIC values between 0.31-0.63 mg/mL. Tis broad-spectrum activity of the acetone extracts indicated that the bioactive extracts were nonselective for the type of microbial cell wall. Te presence of diferent types of bioactive phytochemicals in the acetone extracts such as phenolic compounds, the esters of weak acids, fatty acids, terpenes, and others can afect multiple target sites against the bacterial cells [57].
Only the acetone extracts of G. volkensii and C. glabrum showed inhibitory activity against M. tuberculosis at 0.63 and 2.5 mg/mL, respectively. Our results were consistent with previous studies with slight variations where C. glabrum was reported to have a MIC of 0.156 mg/mL and 0.312 mg/mL against M. tuberculosis and M. smegmatis, respectively [58]. Although ethnobotanical studies report G. volkensii to be used traditionally for tuberculosis treatment [20], the antimycobacterial activity of this plant has not yet been reported. Te antimycobacterial activities reported in this study provide scientifc credence to the anti-TB potential of G. volkensii leaves. Acetone has been reported to extract various hydrophilic and lipophilic components from plant material [59] and may refect a higher probability of diffusing through the mycobacterial cell wall.
Despite belonging to the same family, S. petersiana and A. senegal showed diferent antibacterial properties against the tested microorganisms. All S. petersiana extracts showed signifcant activity against S. aureus while none of the A. senegal extracts was active. It has previously been suggested that MIC values of plant extracts can vary due to the diferences in the present bioactive compounds and the volatile nature of the compounds [60]. Mudi and Salisu [61] reported that the hexane fraction of A. senegal stem bark had good activity against the respiratory tract pathogens such as Klebsiella pneumoniae and Streptococcus pneumoniae. In this work, the hexane extract of the leaves of A. senegal showed the best antibacterial activity against P. aeruginosa with an MIC value of 0.16 mg/mL. Tese results suggest that the leaves may be used as a substitute for the use of stem bark or roots of A. senegal to treat bacterial pneumonia. Te use of the leaves may contribute to sustainability in the harvest and conservation of A. senegal.
One of the important parameters in assessing the microbial activity of plant extracts is total activity (TA). TA (mL) is valuable because it takes into consideration not only the antimicrobial activity but also relates it to the amount of compounds extracted from 1 g of plant material. G. volkensii DCM extract had the highest TA against E. coli (308 mL), and its acetone extract had a higher TA against M. smegmatis (160 mL). Te hexane and acetone extracts of S. petersiana had the highest TA against S. aureus (329 mL) and P. aeruginosa (339 mL). Te TA of these extracts indicated that their MICs can be diluted to high volumes without disrupting the antibacterial activity. Tis concept is important when considering further purifcation or isolation of bioactive compounds from crude extracts [62]. It is known that the active compounds in extracts are commonly produced in lower concentrations, and purifcation processes further reduce the concentrations of these compounds [63]. Terefore, plant extracts with high TA values serve as good candidates for the isolation and characterisation of bioactive antibacterial compounds. Te plant extracts showed bactericidal efects on the tested microorganisms, indicating that the extracts not only inhibit growth but can kill the bacterial cells. Te nonselectivity of the plant extracts on the diferent microorganisms prompted toxicity studies because the antimicrobial activity could be due to toxic compounds in the crude extracts. As such, natural products or their derivatives need to be assessed for their safety before consumption is intended.

Cytotoxicity.
Alveolar macrophages are one of the primary phagocytes that are recruited by the host's immune system to clear the air spaces from foreign particles [64]. Vero cells are homologous with human body cells and easy to culture as the use of this cell line has been representative of normal human cells. Te sensitivity of the Vero cells to toxicity allows them to be ideal in vitro models to be used to evaluate cytotoxicity [65].
Te 0.25% DMSO was used to reconstitute the extracts and was also used as a negative control. Te results showed that at the selected concentration of DMSO (0.25%), there was no signifcant efect on the growth of both the Vero cell line and the THP-1 macrophages (p > 0.05). Tis provided certainty that the solvent did not contribute to the observations. It was notable that each extract exerted diferent cytotoxic efects on the THP-1 and vero cell lines with varying degrees of signifcance. At the lowest tested concentration (100 μg/mL), all the extracts did not signifcantly reduce the cell viability of the THP-1 macrophages (p > 0.05); however, A. senegal, G. volkensii, S. petersiana, and C. glabrum were toxic to vero cell lines (Figure 1) (p < 0.05). S. petersiana ethanol extracts were reported to be toxic to vervet monkey cells, exhibiting ID 50 values of 24 μg/ mL [66]. A. senegal and G. volkensii extracts were generally the most cytotoxic to the vero cell line at all the tested concentrations. Te toxicity exerted by the extracts on the normal vero cell lines may be due to challenges the cell lines experience in culture such as being modifed by many substances present in the crude extracts [67]. Te  Evidence-Based Complementary and Alternative Medicine 9 dichloromethane and methanol extracts of G. volkensii twigs/bark have been reported to be toxic by using the micronuclei and comet assays [68]. In addition, the fruit extract (methanol and chloroform) of G. volkensii moderately had lethality against brine shrimp (Artemia Salina) [22]. Our results were consistent with these previous toxicological studies on G. volkensii and demonstrated the cytotoxicity of eukaryotic cells. Clerodendrum glabrum acetone extract was the most nontoxic to THP-1 macrophages with a nonsignifcant decrease in viable cells at all tested concentrations. Similarly, C. glabrum acetone leaves extracts were previously reported to be nontoxic to the Vero cell line (357.11 μg/mL LC 50 ) [58]. Te general nontoxicity of C. glabrum on diferent cell lines may explain the lack of toxicity case reports associated with its traditional use. C. bispinosa extract was most toxic to the macrophages, suggesting that the immune system may be negatively afected after 24 h exposure. Te C. bispinosa fruit is popular among locals in KwaZulu-Natal (South Africa) and is commonly sold in the summer season [31]. Te widespread use of the fruit and the lack of acute toxicology efects may imply that it is not being consumed in toxic concentrations. It may be worthwhile to further validate the toxicity evaluated in this study by considering in vivo experiments where the activity is high enough to justify it. Te toxicity of the crude extracts could be due to a toxic compound; thus, the safety/antimicrobial activity can be altered by solvent-solvent fractionation [69].

Anti-Infammatory Activity.
Respiratory tract infections are associated with lung tissue damage where the epithelial cells are the frst to be afected. Te tissue damage can be both from microbial infection and the infammatory response of the host. Macrophages produce large amounts of proinfammatory chemical species in response to danger signals [70]. If the injury persists, proinfammatory signals continue and further damage the epithelium of the surrounding cells. For tissue repair to take place and restore normal tissue, infammation must frst be reduced [70]. Figure 2 represents the bovine serum albumin (BSA) denaturation inhibition of the extracts. As expected, diclofenac sodium showed concentration-dependentin vitro BSA denaturation inhibition [45]. Similarly, the plant extracts demonstrated a concentration-dependent inhibition. It was observed that the hexane extracts had better anti-BSA denaturation activity, followed by methanol and water. Tis suggested that nonpolar compounds had better antiinfammatory activity than polar compounds. At 0.25 and 0.5 mg/mL concentrations, hexane extracts from C. bispinosa, and G. volkensii demonstrated comparable antiinfammatory activity to diclofenac sodium (Figure 2). G. volkensii had the highest anti-BSA denaturation activity among the tested plant species. No notable antiinfammatory studies have previously been reported for C. bispinosa and G. volkensii leaves extracts. For the former, more research has been conducted into other members of the Carissa genus such as Carissa caranda [71] and Carissa macrocarpa [72]. Te traditional use of G. volkensii in the treatment of infammation-associated diseases and conditions has been reported [20] and may explain the observed antiinfammatory activities. Only polar solvents, methanol, and water extracts demonstrated antiegg albumin denaturation activity (Figure 3). S. petersiana water extract showed good antiegg albumin denaturation activity (>60% inhibition). Similarly, Aremu et al. [73] reported 99.3 ± 1.2% inhibition of cyclooxygenase 1 (COX-1) by S. petersiana leaves water extract. Te albumin assays demonstrated that the plant extracts have diferent mechanisms of antiinfammatory action and may help reduce the negative impact of respiratory diseases which are also characterized by an increase in and prolonged infammation. In addition, plant extracts with antiprotein denaturation activities have been shown to exhibit thrombolytic and antinociceptive potential in animal models [74,75].

Antioxidant
Activity. DPPH radical scavenging activity and ferric-reducing power expressed as EC 50 values are presented in Table 3. Low EC 50 values indicated that a small amount of the extract is required to reduce half of the total amount of the free radical in a solution. Te water extracts of G. volkensii, S. petersiana, and A. senegal showed the highest DPPH radical scavenging activity for each plant. In support, water extracts have been reported to possess antioxidant activity because of the high presence of phenolic chemicals [76]. Polyphenolic compounds are associated with antioxidant activity due to their ability to readily donate hydrogen and/or electrons. Te highest DPPH radical scavenging activity of all the plant extracts was the C. bispinosa hexane extract, indicating that nonpolar phytochemicals in the extract had strong radical scavenging potential (EC 50 160.65 ± 0.21 μg/mL). Terpenes have been reported to possess antioxidant activity [77], and this may explain the presence of antioxidant activity in the hexane extract of C. bispinosa. Moderate DPPH radical scavenging activity of extracts from G. volkensii fruit has previously been reported [22], and favonoids from the fruit of C. bispinosa have been shown to have moderate DPPH radical scavenging activity [32]. Terefore, our antioxidant study suggests that the leaves of C. bispinosa and G. volkensii are better sources of antioxidant compounds than their fruits. Te acetone leaf extract of G. volkensii had the greatest ferric-reducing power (123.64 ± 0.41 μg/mL). Ascorbic acid had higher free radical scavenging activity (20.41 ± 0.92 μg/mL) and ferric-reducing power (22.67 ± 1.68 μg/mL) than all the tested extracts. Te beneft of the reducing capability of the plant extracts is the reduction of the efects of oxidative stress such as cell and tissue damage which can lead to gradual weakness of the immune system. Tis may further rationalize the traditional use of these selected plant species for not only the treatment of respiratory tract infections but also the treatment of associated symptoms such as fever, cough, and chest pains.

Phytochemical Analysis.
Due to the polarity of phenolics, it was expected that total phenolic content would likely be the highest in the polar solvents. Indeed, phenolic content increased from the hexane, dichloromethane,    (Table 4). Te water extracts of S. petersiana had the highest total phenolic content (TPC) of 1239.94 ± 0.18 mg GAE/g and total tannin content of 14.14 ± 0.24 mg GAE/g (p < 0.05). A favonoid, luteolin, was previously isolated from the ethanol seed extract of S. petersiana [66]. Te lowest TPC was detected in the DCM extract of C. bispinosa (14.70 ± 0.14 mg GAE/g) followed by A. senegal methanolic extract (17.06 ± 0.35 mg GAE/g). Te fruit extracts of C. bispinosa have also been reported to possess bioactive favonoids [32]. A. senegal water extract possessed significantly higher total proanthocyanidin content (579.54 ± 0.32 mg GAE/g). Similarly, favonoids, alkaloids, and tannins in the acetone and methanol leaves extracts of A. senegal have previously been detected [78]. Te hexane and dichloromethane extracts of G. volkensii had the lowest proanthocyanidin and tannin content. Te results obtained from the investigation of phytochemical content give preliminary data on some important groups that are present in the plants investigated. Te phytochemical analysis not only demonstrated the presence of high quantities of bioactive phytochemicals but also showed that the crude extracts indeed consist of a myriad of compounds. Further analysis is required for the identifcation of specifc bioactive compounds to enable a detailed elucidation of the possible mechanisms of action.  (Table 5). Gammalinolenate is an essential polyunsaturated fatty acid and the frst intermediate in the bioconversion of linolenic acid to the long-chain polyunsaturated fatty acid, arachidonic acid [79]. GLA defciency has been associated with diferent physiologic/pathophysiologic conditions [80]. Te role of Gamma-linolenate in fatty acid metabolism may explain its presence in S. petersiana, A. senegal, G. volkensii, and C. bispinosa and may suggest their nutraceutical potential. Indeed, the nutraceutical potential of the carissa genus has been reported [29]. Cetylpyridinium was detected in A. senegal and C. bispinosa at 8.47 and 8.50 mins RT. Te mechanisms of action of cetylpyridinium have been reported to involve interference with osmoregulation and homeostasis of the bacterial cells, and high concentrations lead to degeneration of the membranes with subsequent leakage of cytoplasmic contents [81].
Previous phytochemical studies of G. volkensii leaves reported the presence of various diterpenes and triterpenoids in the crude hexane leaves extracts [82]. In this work, a pentacyclic triterpenoid, cochalate was detected in G. volkensii acetone leaves extract with an 11.78 min RT. Consistently, the seed and the pulp of G. volkensii have been reported to possess triterpenes [22]. Te broad-spectrum antibacterial activities demonstrated by G. volkensii leaves extracts may be attributed to the presence of various bioactive terpenes and terpenoids extracted with acetone and hexane. Numerous polyphenolic compounds were detected in the C. glabrum acetone extract. In this study, two favonoids, 7-hydroxy-2-(4-methoxyphenyl)-4-oxo-       Evidence-Based Complementary and Alternative Medicine chroman-5-olate and (3R)-3-(2,4-dimethoxyphenyl)-7hydroxy-4-oxo-chroman-5-olate were detected at 8.20 and 8.43 mins RT, respectively. Moreover, pentanoic acid (valeric acid) was detected at 5.05 min RT. Valeric acid has been shown to be active against Gram-negative and Grampositive microorganisms [83]. 3,5-Dimethoxy-benzoic acid was isolated from the leaves of C. glabrum cultivated in Egypt [33]. Due to their lipophilic properties, carboxylic acids and carboxylate compounds have been reported to have notable antimycobacterial activity [84]. Te presence of these phytochemicals in the C. glabrum acetone extract may be contributing to the determined antimycobacterial activity.

Conclusions
From our research work, we can conclude that the selected medicinal plants demonstrated their potential use as a phytotherapeutic agent toward the treatment of bacterial respiratory tract infections. Notably, the acetone extracts of G. volkensii and C. glabrum had antimycobacterial activity against M. tuberculosis H37Rv. Our results showed that the extracts had anti-infammatory and antioxidant activities. It is important that the knowledge be documented and the traditional use be given some credence through modern scientifc studies. By using this study, the traditional application of the leaves of the plants as remedies toward bacterial infections may be favoured/approved. Te isolation of the bioactive compounds and characterisation of the specifc mechanisms of action responsible for the observed antibacterial and antiinfammatory activities still require more analysis. Moreover, the use of animal models is required to further validate the anti-TB and antinociceptive potential of the extracts.

Data Availability
Te data used to support the fndings of this study are available from the corresponding authors upon reasonable request.

Ethical Approval
Te study was ethically cleared by the Turfoop Research and Ethics Committee (TREC/80/2021: PG).

Conflicts of Interest
Te authors declare that there are no potential conficts of interest.

Authors' Contributions
MMM carried out the experiments, analysed the data, and drafted of the manuscript. GTM performed toxicological studies, and PM was responsible for study design, data collection, analysis and interpretation of data, and drafting of the manuscript. All authors read and approved the fnal manuscript.